Simvastatin suppresses tissue factor expression and increases fibrinolytic activity in tumor necrosis factor-α–activated human peritoneal mesothelial cells  Bettina Haslinger, Robert Kleemann, Karin H. Toet, Teake Kooistra  Kidney International  Volume 63, Issue 6, Pages 2065-2074 (June 2003) DOI: 10.1046/j.1523-1755.2003.t01-2-00004.x Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 1 Time- and concentration-dependent effect of simvastatin on tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) production in tumor necrosis factor-α (TNF-α)–activated human peritoneal mesothelial cells (HMC). HMC were incubated for two consecutive periods of 24 hours with TNF-α (50 U/mL) (■) or without (□), and with different concentrations of simvastatin. The conditioned media were analyzed for t-PA (A and B) and PAI-1 (C and D) antigens. Results shown are production levels over the first (A and C) and second (B and D) 24-hour incubation period and are mean ± SD of three independent experiments performed in triplicate. *Significant difference (P < 0.05) with corresponding control values. Kidney International 2003 63, 2065-2074DOI: (10.1046/j.1523-1755.2003.t01-2-00004.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 2 Effect of simvastatin on tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) production in human peritoneal mesothelial cells (HMC) pretreated with tumor necrosis factor-α (TNF-α). HMC were incubated for three consecutive periods of 24 hours with TNF-α (10 U/mL) (■), or without (□), while simvastatin (1 μmol/L) was present only during the second and third incubation period. The conditioned media were analyzed for t-PA (A) and PAI-1 (B) antigens. Results shown are production levels over the third 24-hour incubation period and are mean ± SD of three independent experiments performed in triplicate. *Significant difference (P < 0.05) with corresponding control values. Kidney International 2003 63, 2065-2074DOI: (10.1046/j.1523-1755.2003.t01-2-00004.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 3 Time-course of tissue factor (TF) expression in tumor necrosis factor-α (TNF-α)–stimulated human peritoneal mesothelial cells (HMC) incubated with or without simvastatin (Sim). HMC were incubated for 24 hours and 48 hours with (+) or without (-) 1 μmol/L simvastatin for TF mRNA analysis. One hour before the end of the incubation TNF-α (10 U/mL) (+) or phosphate-buffered saline (PBS) (-) was added to the cells. Total RNA was isolated and analyzed by Northern blot hybridization for TF and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The experiment shown is representative for three independent experiments (A). The signals for TF mRNA were quantified by densitometry and adjusted for corresponding GAPDH mRNA signals. The amount of TF mRNA at the different time points is expressed relative to that found at t = 0 (B). The results shown in (B) are means ± SD of three independent experiments. Symbols are: (■) +TNF-α; (□) -TNF-α. *Significant difference (P < 0.05) with control values at t = 0. Kidney International 2003 63, 2065-2074DOI: (10.1046/j.1523-1755.2003.t01-2-00004.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 4 Effect of increasing concentrations of simvastatin (Sim) on tissue factor (TF) expression in tumor necrosis factor-α (TNF-α)–stimulated human peritoneal mesothelial cells (HMC). HMC were incubated for 48 hours with increasing concentrations of simvastatin for mRNA analysis. One hour before the end of the incubation, TNF-α (10 U/mL) (+) or phosphate-buffered saline (PBS) (-) was added to the cells. Total RNA was isolated and analyzed by Northern blot hybridization for TF and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The experiment shown is representative for three independent experiments (A). The signals for TF mRNA were quantified by densitometry and adjusted for corresponding GAPDH mRNA signals. The amount of TF mRNA at the different simvastatin concentrations is expressed relative to that found at control conditions (B). The results shown in (B) are means ± SD of three independent experiments. Symbols are: (■) +TNF-α; (□) -TNF-α. *Significant difference (P < 0.05) with corresponding control values. Kidney International 2003 63, 2065-2074DOI: (10.1046/j.1523-1755.2003.t01-2-00004.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 5 Inhibition of the simvastatin (Sim) effect on tissue factor (TF) expression by mevalonic acid (MVA) and geranylgeraniol (GG) in human peritoneal mesothelial cells (HMC). HMC were incubated for 48 hours with (+) or without (-) 1 μmol/L simvastatin in the presence or absence of 100 μmol/L MVA or 10 μmol/L GG for TF mRNA analysis. One hour before the end of the incubation, tumor necrosis factor-α (TNF-α) (10 U/mL) (+) or phosphate-buffered saline (PBS) (-) was added to the cells. Total RNA was isolated and analyzed by Northern blot hybridization for TF and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The experiment shown is representative for three independent experiments (A). The signals for TF mRNA were quantified by densitometry and adjusted for corresponding GAPDH mRNA signals. The amount of TF mRNA under the different experimental conditions is expressed relative to that found under control conditions. The results shown in (B) are means ± SD of three independent experiments. Symbols are: (■) +TNF-α; (□) -TNF-α. *Significant difference (P < 0.05) with control values. Kidney International 2003 63, 2065-2074DOI: (10.1046/j.1523-1755.2003.t01-2-00004.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 6 The effect of simvastatin on the activation of nuclear factor kappa B (NF-κB). (A) HT1080 fibrosarcoma cells were pretreated for 24 hours with simvastatin (0.3, 1, or 3 μmol/L) before transient transfection with a 3 × NF-κB-luciferase reporter plasmid. After transfection, the cells were incubated for an additional 24 hours with simvastatin (0.3, 1, 3 μmol/L) and for 17 hours with tumor necrosis factor-α (TNF-α) (100 U/mL) (+) or phosphate-buffered saline (PBS) (-). Results are given as means ± SD of three independent experiments. Symbols are: (■) +TNF-α; (□) -TNF-α. *P < 0.05 compared with control. (B) HMC were incubated for 48 hours with (+) or without (-) 1 μmol/L simvastatin (Sim). One hour before the end of the incubation period, tumor necrosis factor-α (TNF-α) (10 U/mL) (+) or phosphate-buffered saline (PBS) (-) was added to the cells. Nuclear extracts were prepared and analyzed for p50–NF-κB and p65–NF-κB expression by Western blotting. Histone H1 levels are shown as a control. The experiment shown is representative for three independent experiments. Kidney International 2003 63, 2065-2074DOI: (10.1046/j.1523-1755.2003.t01-2-00004.x) Copyright © 2003 International Society of Nephrology Terms and Conditions

Figure 7 The effect of simvastatin on the activation of activator protein-1 (AP-1). (A) HT1080 fibrosarcoma cells were pretreated for 24 hours with simvastatin (Sim) (0.3, 1, or 3 μmol/L) before transient transfection with a collagenase-5 × 12-o-tetradecanol phorbol acetate (TRE)-luciferase reporter plasmid. After transfection, the cells were incubated for an additional 24 hours with simvastatin (0.3, 1, 3 μmol/L) and for 17 hours with tumor necrosis factor-α (TNF-α) (100 U/mL) (+) or phosphate-buffered saline (PBS) (-). Results are given as means ± SD of three independent experiments. Symbols are: (■) +TNF-α; (□) -TNF-α. *P < 0.05 compared with control. (B) Human peritoneal mesothelial cells (HMC) were incubated for 48 hours with (+) or without (-) 1 μmol/L simvastatin; 1 hour before the end of the incubation, TNF-α (10 U/mL) (+) or PBS (-) was added to the cells. Nuclear extracts were prepared and analyzed for c-jun and c-fos expression by Western blotting. Histone H1 levels are shown as a control. The experiment shown is representative for three independent experiments. Kidney International 2003 63, 2065-2074DOI: (10.1046/j.1523-1755.2003.t01-2-00004.x) Copyright © 2003 International Society of Nephrology Terms and Conditions